Project Summary Spinal cord injury (SCI) is among the most disabling conditions affecting wounded members of the U.S. military. Unfortunately, no effective treatment has been available for patients with SCI. Developing novel repair strategies to mitigate the devastating nature of SCI and translating them to the clinic are urgent medical needs for our veterans with SCI. For functional recovery to occur after a SCI, regenerated axons need to follow the topography of grafted cells/growth factors and make accurate connections with specific subsets of neurons or subregions of dendritic architecture. The lumbar motoneurons (MNs) are the final common pathways for motor output to the hindlimbs and they undergo dendritic atrophy and synaptic stripping after an above-level SCI. The goal of our research is to reestablish neural circuitry across the lesion gap and to promote functional recovery after SCI. We hypothesize that a growth promoting pathway composed of grafted Schwann cells (SCs) overexpressing a growth factor called glial cell line-derived neurotrophic factor (GDNF) will promote the growth of descending propriospinal tract (dPST) axons across the lesion gap with extension caudally to the lumbar MNs in the host spinal cord, and that these axons will form target-specific synaptic contacts with lumbar MNs overexpressing a neurotrophin called neurotrophin-3 (NT-3). We also hypothesize that such a combinatorial approach will lead to greater recovery of function than either single treatment. Using a clinically-relevant contusive SCI model at the 9th thoracic (T9) level, transplantation of SCs-GDNF to form a continuous axonal growth-promoting pathway across and beyond a SCI, and adeno-associated virus serotype 2 expressing NT-3 (AAV2-NT-3) gene transfer approach to enhance NT-3 expression in lumbar MNs, we will determine (1) whether a continuous axonal growth-promoting pathway formed by grafted SCs-GDNF will promote dPST axonal growth through and beyond a contusive SCI, innervate the lumbar MNs pools, and enhance electrophysiological and locomotor recoveries; (2) whether combining the axonal growth-promoting pathway formed by SCs-GDNF with expression of NT-3 in lumbar MNs will synergistically enhance the innervation of dPST axons on lumbar MNs and, therefore, promote better recovery of function as compared to either treatment alone; (3) whether dPST-MN neurotransmission is necessary for hindlimb locomotor recovery in the combinatorial treatment; and (4) the molecular signature of lumbar MNs after the reestablishment of dPST-MN circuitry and synaptogenesis. Completion of this proposal will allow us to reveal mechanisms fundamental to rebuilding neural circuitry of the dPST-MN pathway and to identify new therapeutic strategies for locomotor recovery after clinically-relevant contusive SCIs.